Protein Inhibitor of Neuronal Nitric Oxide Synthase (PIN) Is a New Regulator of Glucose-Induced Insulin Secretion

Abstract

We previously showed that pancreatic ␤-cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now provide evidence that the endogenous protein inhibitor of nNOS (PIN) is expressed in rat pancreatic islets and INS-1 cells. Double-immunofluorescence studies showed a colocalization of PIN with both nNOS and myosin Va in insulin-secreting ␤-cells. Electron microscopy studies confirmed that PIN is mainly associated with insulin secretory granules and colocated with nNOS in the latter. In addition, PIN overexpression in INS-1 cells enhanced glucoseinduced insulin secretion, which is only partly reversed by addition of an NO donor, sodium nitroprusside (SNP), and unaffected by the inhibitor of cytochrome c reductase activity, miconazole. In contrast, the pharmacological inhibitor of nNOS, N-nitro-L-arginine methyl ester, amplified glucose-induced insulin secretion, an effect insensitive to SNP but completely normalized by the addition of miconazole. Thus, PIN insulinotropic effect could be related to its colocalization with the actin-based molecular motor myosin Va and as such be implicated in the physiological regulation of glucose-induced insulin secretion at the level of the exocytotic machinery. Diabetes 55: 3279 -3288, 2006 T he second messenger nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as NO synthases (NOSs). Three NOS isoforms have been identified, including the constitutive neuronal NO synthase (nNOS), endothelial NOS (eNOS), and the cytokine-inducible NOS (iNOS) (1). Constitutive NOSs produce small amounts of NO, implicated in physiological functions such as synaptic transmission (2) and vascular tone (3), whereas iNOS produces high amounts of NO, acting as a cytotoxic mediator in immune response (4). In addition to these beneficial roles, NO exerts deleterious effects as a result of its overproduction, leading to neuronal death during brain ischemia and certain neurodegenerative pathologies (5) and to autoimmune diseases (6). For these reasons, a tight regulation of NOS catalytic activity is essential for its physiological function. Modulation of iNOS expression by cytokines is believed to be the most important component of iNOS regulation, but posttranscriptional mechanisms also operate (7,8). As for the constitutive isoforms, catalytic activity is closely modulated by different mechanisms. The enzyme's activity is first controlled by the reversible binding of Ca 2ϩ -calmodulin (9), which allows the transfer of NADPH-derived electrons from the reductase to the oxygenase domain of the enzyme (10). It is also positively or negatively regulated through phosphorylation by various protein kinases such as cAMP-dependent protein kinase and protein kinase C (11,12). In addition to the role of intracellular Ca 2ϩ levels and protein kinases, constitutive NOSs are also regulated via protein-protein interactions. Thus, eNOS-associated protein-1 (ENAP-1...

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

“…From a pathophysiological point of view, alterations in the nNOS signalling pathway resulting from nNOS mislocalisation might be involved in muscle fatigue and atrophy intervening in insulin-resistant skeletal muscle [45,46]. Concerning the expression of the protein inhibitor of nNOS, PIN, originally cloned in rat brain [18], and identified in ventilatory muscles [43] and pancreatic beta cells [47], we found levels of PIN to be almost unchanged in insulin-resistant muscles. In addition, PIN sarcolemmal localisation decreased in line with that of nNOS, pointing to a minor role of PIN in nNOS alterations.…”

Increased neuronal nitric oxide synthase dimerisation is involved in rat and human pancreatic beta cell hyperactivity in obesity

“…Indeed, we have previously shown by electron microscopy that nNOS was present at the level of insulin granules in rat beta cells [10], where the enzyme interacts with its endogenous protein inhibitor, protein inhibitor of neuronal NOS (PIN) [31]. PIN is a highly conserved protein that blocks the enzyme dimerisation and subsequent NO production [32].…”

Neuronal Nitric Oxide Synthase

“…In contrast, nNOS inhibitor increased insulin secretion induced by glucose. Therefore, PIN insulinotropic effect could be related to its co-localization with the actin-based molecular motor myosin and as such be involved in the physiological regulation of insulin secretion at the exocytotic machinery [33].…”

“…Pancreatic β-cells express nNOS that controls insulin secretion through two catalytic activities: nitric oxide production and cytochrome c reductase activity [33].…”